Preiss Rainer, Schmidt Renate, Baumann Frank, Hanschmann Henning, Hauss Johann, Geissler Felix, Pahlig Hartmut, Ratzewiss Bernd
Institute of Clinical Pharmacology, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany.
J Cancer Res Clin Oncol. 2002 Jul;128(7):385-92. doi: 10.1007/s00432-002-0335-4. Epub 2002 Jun 11.
The aim of the present study was to determine the turnover (4-hydroxylation and N-dechloroethylation) of ifosfamide in a total of 25 human liver microsomal preparations in which the codetermination of keto- and carboxyifosfamide as well as the calculation of free and protein-bound acrolein was carried out for the first time.
The 4-hydroxylation of ifosfamide was estimated by using acrolein (free and protein-bound) and a newly developed procedure involving the codetermination of keto- and carboxyifosfamide (LC/MS). The ifosfamide N-dechloroethylation was determined as the sum of 2- and 3-dechloroethylifosfamide (LC/MS).
Using the usual estimation of liberated free acrolein in 25 human liver microsomal preparations, the 4-hydroxylation of ifosfamide amounted to 0.28+/-0.16 nmol/min. nmol(P450). However, after calculating the 4-hydroxylation as the sum of free and protein-bound acrolein and keto- and carboxyifosfamide, a ninefold higher activity (2.40+/-0.73 nmol/min. nmol(P450)) was found. The percentage of the inactive metabolites keto- (25/25) and carboxyifosfamide (5/25) in the 4-hydroxylation amounted to only 0.79-5.25% (mean 2.90%). The ifosfamide N-dechloroethylation (mean 0.21+/-0.11 nmol/min. nmol(P450)) determined as the sum of 2- and 3-dechloroethylifosfamide was estimated as 8.3+/-4.3% of the total ifosfamide turnover. The application of the relative substrate-activity factor (RSF)-approach and the calculation of the contribution of various isoforms in the ifosfamide 4-hydroxylation yielded the following results: CYP 3A4: 58+/-31%, CYP 2A6: 25+/-15%, and CYP 2C9: 5+/-2% of the total measured 4-hydroxylation. A correlation between 4-hydroxylation and the N-dechloroethylation rates of ifosfamide and the activities of isoenzymes indicates the involvement of both CYP 3A4 ( P=0.026) and CYP 2C9 ( P=0.012) in the 4-hydroxylation reaction and of CYP 3A4 ( P<0.01) in the N-dechloroethylation reaction.
The estimation of protein-bound acrolein should be included in the calculation of the ifosfamide 4-hydroxylation besides liberated free acrolein. Because of the small amounts of the inactive metabolites keto- and carboxyifosfamide, the exclusive determination of acrolein only (free and protein-bound) seems to suffice for the calculation of total ifosfamide hydroxylation. Using this method the hepatic in vitro turnover of ifosfamide was estimated as 92% for 4-hydroxylation (CYP 3A4 and CYP 2A6 mediated) and 8% for N-dechloroethylation (CYP 3A4 mediated), and in this way, a relative overestimation of the N-dechloroethylation of ifosfamide on the whole metabolism is avoided.
本研究旨在测定异环磷酰胺在总共25种人肝微粒体制剂中的代谢转化（4-羟基化和N-脱氯乙基化），首次对酮异环磷酰胺和羧基异环磷酰胺进行了共同测定，并计算了游离和与蛋白结合的丙烯醛。
通过使用丙烯醛（游离和与蛋白结合的）以及一种新开发的涉及酮异环磷酰胺和羧基异环磷酰胺共同测定的方法（液相色谱/质谱法）来评估异环磷酰胺的4-羟基化。将异环磷酰胺N-脱氯乙基化测定为2-脱氯乙基异环磷酰胺和3-脱氯乙基异环磷酰胺的总和（液相色谱/质谱法）。
在25种人肝微粒体制剂中，按照常规方法估算游离释放的丙烯醛，异环磷酰胺的4-羟基化量为0.28±0.16 nmol/分钟·nmol（细胞色素P450）。然而，在将4-羟基化计算为游离和与蛋白结合的丙烯醛以及酮异环磷酰胺和羧基异环磷酰胺的总和后，发现活性提高了九倍（2.40±0.73 nmol/分钟·nmol（细胞色素P450））。在4-羟基化过程中，无活性代谢产物酮异环磷酰胺（25/25）和羧基异环磷酰胺（5/25）的百分比仅为0.79 - 5.25%（平均2.90%）。将异环磷酰胺N-脱氯乙基化（平均0.21±0.11 nmol/分钟·nmol（细胞色素P450））测定为2-脱氯乙基异环磷酰胺和3-脱氯乙基异环磷酰胺的总和，估计占异环磷酰胺总代谢转化的8.3±4.3%。应用相对底物活性因子（RSF）方法并计算异环磷酰胺4-羟基化中各种同工型的贡献得出以下结果：细胞色素P450 3A4：占总测定4-羟基化的58±31%，细胞色素P450 2A6：25±15%，细胞色素P450 2C9：5±2%。异环磷酰胺的4-羟基化和N-脱氯乙基化速率与同工酶活性之间的相关性表明，细胞色素P450 3A4（P = 0.026）和细胞色素P450 2C9（P = 0.012）参与4-羟基化反应，细胞色素P450 3A4（P < 0.01）参与N-脱氯乙基化反应。
在计算异环磷酰胺4-羟基化时，除了游离释放的丙烯醛外，还应包括与蛋白结合的丙烯醛的估算。由于酮异环磷酰胺和羧基异环磷酰胺等无活性代谢产物的量较少，仅单独测定丙烯醛（游离和与蛋白结合的）似乎就足以计算异环磷酰胺的总羟基化。使用这种方法，异环磷酰胺的肝体外代谢转化估计为4-羟基化（由细胞色素P450 3A4和细胞色素P450 2A6介导）占92%，N-脱氯乙基化（由细胞色素P450 3A4介导）占8%，这样就避免了在整个代谢过程中对异环磷酰胺N-脱氯乙基化的相对高估。
